The present invention relates to the production and screening of expression libraries for enzyme activity and, more particularly, to obtaining selected polynucleotides from nucleic acid of a microorganism and to screening of an expression library for enzyme activity which is produced from selected polynucleotides.
There is a critical need in the chemical industry for efficient catalysts for the practical synthesis of optically pure materials; enzymes can provide the optimal solution. All classes of molecules and compounds that are utilized in both established and emerging chemical, pharmaceutical, textile, food and feed, detergent markets must meet stringent economical and environmental standards. The synthesis of polymers, pharmaceuticals, natural products and agrochemicals is often hampered by expensive processes which produce harmful byproducts and which suffer from low enantioselectivity. Enzymes have a number of remarkable advantages which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, selectivity, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries. Enzyme-based processes have been gradually replacing many conventional chemical-based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes. Only xcx9c300 enzymes (excluding DNA modifying enzymes) are at present commercially available from the  greater than 3000 non DNA-modifying enzyme activities thus far described.
The use of enzymes for technological applications also may require performance under demanding industrial conditions. This includes activities in environments or on substrates for which the currently known arsenal of enzymes was not evolutionarily selected. Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of mild temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far described have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments. Such enzymes must function at temperatures above 100xc2x0 C. in terrestrial hot springs and deep sea thermal vents, at temperatures below 0xc2x0 C. in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge. Enzymes obtained from these extremophilic organisms open a new field in biocatalysis.
In addition to the need for new enzymes for industrial use, there has been a dramatic increase in the need for bioactive compounds with novel activities. This demand has arisen largely from changes in worldwide demographics coupled with the clear and increasing trend in the number of pathogenic organisms that are resistant to currently available antibiotics. For example, while there has been a surge in demand for antibacterial drugs in emerging nations with young populations, countries with aging populations, such as the U.S., require a growing repertoire of drugs against cancer, diabetes, arthritis and other debilitating conditions. The death rate from infectious diseases has increased 58% between 1980 and 1992 and it has been estimated that the emergence of antibiotic resistant microbes has added in excess of $30 billion annually to the cost of health care in the U.S. alone. (Adams et al., Chemical and Engineering News, 1995; Amann et al., Microbiological Reviews, 59, 1995). As a response to this trend pharmaceutical companies have significantly increased their screening of microbial diversity for compounds with unique activities or specificities.
There are several common sources of lead compounds (drug candidates), including natural product collections, synthetic chemical collections, and synthetic combinatorial chemical libraries, such as nucleotides, peptides, or other polymeric molecules. Each of these sources has advantages and disadvantages. The success of programs to screen these candidates depends largely on the number of compounds entering the programs, and pharmaceutical companies have to date screened hundred of thousands of synthetic and natural compounds in search of lead compounds. Unfortunately, the ratio of novel to previously-discovered compounds has diminished with time. The discovery rate of novel lead compounds has not kept pace with demand despite the best efforts of pharmaceutical companies. There exists a strong need for accessing new sources of potential drug candidates.
The majority of bioactive compounds currently in use are derived from soil microorganisms. Many microbes inhabiting soils and other complex ecological communities produce a variety of compounds that increase their ability to survive and proliferate. These compounds are generally thought to be nonessential for growth of the organism and are synthesized with the aid of genes involved in intermediary metabolism hence their name xe2x80x9csecondary metabolitesxe2x80x9d. Secondary metabolites that influence the growth or survival of other organisms are known as xe2x80x9cbioactivexe2x80x9d compounds and serve as key components of the chemical defense arsenal of both micro- and macroorganisms. Humans have exploited these compounds for use as antibiotics, antiinfectives and other bioactive compounds with activity against a broad range of prokaryotic and eukaryotic pathogens. Approximately 6,000 bioactive compounds of microbial origin have been characterized, with more than 60% produced by the gram positive soil bacteria of the genus Streptomyces. (Barnes et al., Proc. Nat. Acad Sci. U.S.A., 91, 1994). Of these, at least 70 are currently used for biomedical and agricultural applications. The largest class of bioactive compounds, the polyketides, include a broad range of antibiotics, immunosuppressants and anticancer agents which together account for sales of over $5 billion per year.
Despite the seemingly large number of available bioactive compounds, it is clear that one of the greatest challenges facing modern biomedical science is the proliferation of antibiotic resistant pathogens. Because of their short generation time and ability to readily exchange genetic information, pathogenic microbes have rapidly evolved and disseminated resistance mechanisms against virtually all classes of antibiotic compounds. For example, there are virulent strains of the human pathogens Staphylococcus and Streptococcus that can now be treated with but a single antibiotic, vancomycin, and resistance to this compound will require only the transfer of a single gene, vana, from resistant Enterococcus species for this to occur. (Bateson et al., System. Appl. Microbiol, 12, 1989). When this crucial need for novel antibacterial compounds is superimposed on the growing demand for enzyme inhibitors, immunosuppressants and anti-cancer agents it becomes readily apparent why pharmaceutical companies have stepped up their screening of microbial diversity for bioactive compounds with novel properties.
The present invention provides a novel approach for obtaining enzymes for further use, for example, for a wide variety of industrial applications, for medical applications, for packaging into kits for use as research reagents and for other applications. In accordance with the present invention, recombinant enzymes are generated from microorganisms and are classified by various enzyme characteristics.
More particularly, one aspect of the present invention provides a process for identifying clones having a specified enzyme activity, which process comprises screening for said specified enzyme activity in a library of clones prepared by:
(i) selectively isolating target RNA or genomic DNA or fragments thereof, from nucleic acid derived from at least one microorganism, by use of at least one probe polynucleotide comprising at least a portion of a polynucleotide sequence encoding an enzyme having the specified enzyme activity; and
(ii) transforming a host with isolated target cDNA, genomic DNA or fragments thereof, to produce a library of clones which are screened, preferably for the specified enzyme activity, using an activity library screening or nucleic acid library screening protocol.
In a preferred embodiment of this aspect, nucleic acid obtained from at least one microorganism is selected by recovering from the nucleic acid, polynucleotides which specifically bind, such as by hybridization, to a probe polynucleotide sequence. The nucleic acid obtained from the microorganism or microorganisms can be genomic DNA, RNA or genomic gene library DNA. One could even use nucleic acid prepared for vector ligation, for instance. The probe may be directly or indirectly bound to a solid phase by which it is separated from the nucleic acid which is not hybridized or otherwise specifically bound to the probe. The process can also include releasing nucleic acid from said probe after recovering said hybridized or otherwise bound nucleic acid and amplifying the nucleic acid so released.
The invention also provides for screening of the expression libraries for gene cluster protein product(s) and, more particularly, to obtaining selected gene clusters from nucleic acid of a prokaryote or eukaryote and to screening of an expression library for a desired activity of a protein of related activity(ies) of a family of proteins which results from expression of the selected gene cluster nucleic acid of interest.
More particularly, one embodiment of this aspect provides a process for identifying clones having a specified protein(s) activity, which process comprises screening for said specified enzyme activity in the library of clones prepared by (i) selectively isolating target gene cluster nucleic acid, from nucleic acid derived from at least one organism, by use of at least one probe polynucleotide comprising at least a portion of a polynucleotide sequence complementary to a nucleic acid sequence encoding the protein(s) having the specified activity of interest; and (ii) transforming a host with isolated target gene cluster nucleic acid to produce a library of such clones which are screened for the specified activity of interest. For example, if one is using DNA in a lambda vector one could package the DNA and infect cells via this route.
In a particular embodiment of this aspect, gene cluster nucleic acid obtained from the genomic nucleic acid of the organism(s) is selected by recovering from the nucleic acid, nucleic acid which specifically binds, such as by hybridization, to a probe polynucleotide sequence. The polynucleotide probe may be directly or indirectly bound to a solid phase by which it is separated from the nucleic acid which is not hybridized or otherwise specifically bound to the probe. This embodiment of this aspect of the process of the invention can also include releasing bound nucleic acid from said probe after recovering said hybridized or otherwise bound nucleic acid and amplifying the nucleic acid so released.
These and other aspects of the present invention will be apparent to those skilled in the art from the teachings herein.